This invention relates to a DNA base sequencer, or an apparatus for determining the base sequences of DNA. More particularly, this invention relates to an apparatus with which the base sequences of DNA can be determined by fluorescent labelling in an efficient and rapid manner.
Gel electrophoresis is practiced extensively as a technique for determining the base sequences of DNA and other proteins.
Conventionally, the sample to be subjected to electrophoresis is labelled with a radioisotope for analysis but this method has had the problem of being painstaking and time-consuming. Furthermore, the use of radioactive substances always calls for utmost safety and management and analysis cannot be performed in areas other than facilities that clear certain regulations. Under the circumstances, a method that uses fluorophores to label the sample and which detects fluorescence as emitted upon irradiation with light is being reviewed.
In this method, fluorophore-labelled DNA fragments are caused to migrate through a gel and a light excitation portion and a photodetector are provided for each electrophoresis track in an area 15-20 cm below the start point of electrophoresis. The DNA fragments are assayed as they pass through the line connecting the light excitation portion and the photodetector. A typical procedure of the method is described below. First, using as template the DNA chain to be determined for its base sequence, DNAs of various lengths with known terminal base species are replicated by a method involving an enzymatic reaction (the dideoxy method). Then, the replicated DNAs are labelled with a fluorophore. Stated more specifically, there are prepared a group of adenine (A) fragments, a group of cytosine (C) fragments, a group of guanine (G) fragments and a group of thymine (T) fragments, all being labelled with a fluorophore. A mixture of these fragment groups is injected into separate lane grooves in an electrophoretic gel and, thereafter, a voltage is applied at opposite ends of the gel. Since DNA is a chained polymer with negative charges, it will move across the gel at a rate in inverse proportion to its molecular weight. The shorter the DNA chain (the smaller its molecular weight), the faster will it move and vice versa; this is the principle behind the fractionation of DNA by molecular weight.
Japanese Laid-Open Patent Application (kokai) No. 21556/1988, incorporated herein by reference, teaches a DNA base sequencer that is adapted in such a way that a line on the gel in an apparatus for electrophoresis at which laser light is applied and the direction in which photodiodes are arranged are both perpendicular to the direction in which DNA fragments migrate in the apparatus.
The setup of this apparatus is shown schematically in FIG. 11. In the apparatus shown in FIG. 11, a laser beam emitted from a light source 70 is reflected by a mirror 72 and launched horizontally from one side of the plate 74 at a predetermined point on the gel. As the fluorophore-labelled DNA fragments migrating through the gel pass through the irradiated region, they will fluoresce successively. The horizontal position of fluorescence emission tells the species of a particular terminal base, the time difference from the start of migration tells the length of a particular fragment, and the emission wavelength identifies the sample under assay. The fluorescence from each electrophoresis track is condensed by a lens 78 to focus at a light-receiving area 82 in an image intensifier 80. The received signal is amplified and converted to an electric signal in a photodiode array 84 for the purpose of various measurements. The results of measurements are processed with a computer so that the sequences of the individual DNA fragments are calculated to determine the base sequence of the DNA at issue.
The apparatus shown in FIG. 11 uses an image intensifier camera in the light-receiving optics. The image intensifier camera is not only very expensive but also comparatively large as an optical device. The overall size of the electrophoretic apparatus becomes inevitably bulky.
The light source is a gas laser using Ar or He--Ne as a lasing medium. These laser light sources are usually very expensive. The DNA sequencer of the type that has exciting light applied to the gel layer from one lateral side has the following problems: (1) the beam of exciting laser light cannot be focused to a sufficiently small diameter over a long range so that the spatial resolution is limited; (2) in order to solve this problem, the beam was spread to an elliptical form in the direction of electrophoresis so that the spatial resolution could be improved in the pixels of the sensor; however, most of the beams emitted from gas lasers have a circular cross-sectional profile and complicated illumination optics is required to spread the beam to an elliptical form.
To solve these problems, the present inventors previously invented a DNA base sequencer using a CCD line sensor as light-receiving optics and a laser diode (hereunder LD) as an exciting light source. This apparatus was applied for patent and is now described in Japanese Laid-Open Patent Application (kokai) No. 132784/1998, incorporated herein by reference. The exciting light emitted by LD has a beam divergence angle of 7.5.times.37 degrees. As is apparent from this characteristic value, the laser diode emits a generally elliptical beam. Therefore, unlike a gas laser which emits a beam having a circular cross section, the laser diode does not require any special optics to produce an elliptical beam. Compared to the image intensifier camera, the CCD line sensor is not only very compact but also inexpensive. Similarly, compared to the gas lasers, the LD is not only compact but also very cheap. Therefore, as a result of using the CCD line sensor as light-receiving optics and the LD as an exciting light source, not only the overall size but also the cost of the DNA base sequencer could be dramatically reduced.
However, continued studies of the present inventors have revealed that the oscillation wavelength of LD is instable, making it fairly difficult to obtain a specified wavelength in a consistent manner. As it turned out, LD has the following features: (1) if the optical output is fixed, the forward current increases with increasing temperature; (2) if the temperature of the casing increases, the oscillation wavelength also increases, typically at a rate of 0.23 nm/.degree. C. Under the circumstances, the present inventors made an attempt at holding the oscillation wavelength of LD constant by controlling its temperature with the aid of a Peltier device. The oscillation wavelength of LD also varies with the drive current. To deal with this problem, the present inventors combined LD with a photodiode, detected the current through the photodiode and attempted to keep the LD drive current constant on the basis of the detected current value by means of an automatic power control (APC) device connected to the LD.
In this approach, the LD current and temperature are monitored and one can only presume indirectly that the oscillation wavelength of LD should be constant if the monitored values remain the same. The variations in the oscillation wavelength are not detected. Therefore, even if the temperature of LD casing is controlled to remain constant at 20.degree. C. with the aid of a Peltier device in order to keep the oscillation wavelength of LD at 637 nm, a shift to the longer wavelength side may occur during ID operation. If the operator continues DNA analysis without becoming aware of this event, the data obtained is not completely reliable. If part of the exciting light in the longer wavelength range is received by a fluorescence detector as stray light, it overlaps the fluorescence at a nearby wavelength (say, 650 nm), making it difficult or even impossible to locate or separate the fluorescence that should be detected.
Other studies made on the apparatus shown in FIG. 11 and the apparatus disclosed in Japanese Laid-Open Patent Application No. 132784/1998, supra have revealed the following problems with the use of flat electrophoresis plate: (1) it takes considerable time to inject a fluorophore-labelled DNA sample into all electrophoresis tracks; (2) contamination often occurs due to sample mixing in adjacent electrophoresis tracks; (3) the DNA sample may depart from the correct track to either right or left during electrophoresis and this phenomenon, commonly called "smiling", can cause errors in measurement.
Under the circumstances, a DNA base sequencer was developed that used a hollow capillary, rather than the flat plate, as electrophoretic means. The capillary is filled with a gel electrolyte and a fluorophore-labelled DNA sample is injected into the capillary from one open end; thereafter, the other open end of the capillary is immersed in a buffer tank serving as a negative electrode and the open end from which the DNA sample was injected is immersed in a buffer tank serving as a positive electrode; when a voltage of -15 kV is applied, the DNA fragments are electrophoresed. The apparatus having this construction is disclosed in Japanese Laid-Open Patent Application (kokai) No. 72177/1993, incorporated herein by reference. Specifically, a gas laser is used as an exciting light source and a plurality of capillaries are aligned on a longitudinal axis such that the exciting light travels from one end of the line to the other,
The present inventors made an experiment using as a light source the laser diode of Japanese Laid-Open Patent Application (kokai) No. 132784/1998, supra, instead of the gas laser of Japanese Laid-Open Patent Application (kokai) No. 72177/1993, supra. As it turned out, incident laser light was extensively scattered by the first capillary and the scattered laser light became stray light that was another source of errors in measurement.